Viruses are powerful tools for the development of vaccines and gene therapies. However, the safety of live recombinant vectors is always a concern, as uncontrolled replication or complications are not inconsequential, particularly in immunosuppressed individuals. In an effort to develop safer, yet still effective live viral vectors, we propose to construct fully replicating virus vectors with a safety mechanism designed to be used when complications with the vector occur or therapy needs to be stopped. Our SMART (Safety Mechanism Assisted by the Repressor of Tetracycline) virus vectors will use elements from the tet operon to regulate the expression of a "safety" gene. Vaccinia virus (VV) is an ideal vector system to test this strategy. The tet system has been successfully adapted to VV, allowing expression to be tightly regulated by the antibiotic tetracycline. In addition, we have shown that interferon-gamma (IFN-gamma) acts as a safety gene in vivo when expressed by VV, attenuating the virus by more than million-fold in immunodeficient mice. Our hypothesis is that live SMART VV vectors expressing a safety gene would be significantly safer: treatment of any adverse reactions would be as simple as antibiotic therapy, since it would allow the expression of the safety gene and significantly enhance virus clearance. More importantly, tetracycline treatment would only be needed when complications occur or are suspected, or when treatment should be stopped. Two specific aims will be addressed under this R21 application: (1) to develop SMART VV vectors expressing a safety gene only in the presence of inducer, and (2) to assess the safety and efficacy of the new vectors. First, SMART VV vectors expressing the tetracycline repressor under a constitutive VV promoter and the reporter gene green fluorescent protein (GFP) under an engineered inducible promoter will be generated and the induction of GFP will be examined in an effort to optimize the system. Then, a SMART vector inducibly expressing murine IFN-gamma, as a model safety gene will be developed. Normal and immunodeficient mice will be given the VV vector expressing IFN-gamma inducibly and survival, pock lesion resolution, disease recovery, weight loss, and virus replication will be assessed in the presence and absence of inducer. In addition, immune responses to VV will be assessed to ensure that the efficacy of the new vectors is not compromised by expression tetracycline repressor expression or tetracycline treatment. [unreadable] [unreadable]